Changes in the Blood-Brain Barrier Function Are Associated With Hippocampal Neuron Death in a Kainic Acid Mouse Model of Epilepsy

Abstract

The kainic acid (KA)-induced epilepsy experimental model is widely used to study the mechanisms underlying this disorder. Recently, the blood-brain barrier (BBB) has become an innovative alternative treatment target for epilepsy patients. KA causes neuronal injury and BBB damage in this experimental epilepsy model but the mechanisms underlying epilepsy-related neuronal injury, autophagy, and BBB damage remain unclear. Therefore, the present study investigated the relationships among neuronal injury, the expressions of autophagy-related proteins, and changes in BBB-related proteins during the acute phase of epilepsy to further understand the mechanisms and pharmacotherapy of epilepsy. NeuN immunohistochemistry and Fluoro-Jade B (FJ-B) staining in the hippocampal CA3 region revealed that neuronal death induced by intraventricular injections of 10 μg/kg KA was greater than that induced by 3 μg/kg KA. In addition, there were transient increases in the levels of microtubule-associated protein light chain 3-II (LC3I/II) and Beclin-1, which are autophagy-related proteins involved in neuronal death, in this region 24 h after the administration of 10 μg/kg KA. There were also morphological changes in BBB-related cells such as astrocytes, endothelial cells (ECs), and tight junctions (TJs). More specifically, there was a significant increase in the activation of astrocytes 72 h after the administration of 10 μg/kg KA as well as continuous increases in the expressions of platelet endothelial cell adhesion molecule-1 (PECAM-1) and BBB-related TJ proteins (Zonula occludens-1 and Claudin-5) until 72 h after KA treatment. These results suggest that the overexpression of autophagy-related proteins and astrocytes and transient increases in the expressions of BBB-related TJ proteins may be closely related to autophagic neuronal injury. These findings provide a basis for the identification of novel therapeutic targets for patients with epilepsy.

Keywords: PECAM-1; autophagy related neuronal death; hippocampus CA3; kainic acid; tight junction proteins.

Figure 1

Racine scale. Data represent means ± SEM. ^*P < 0.05 was compared with the control group. ANOVA tests followed by Duncan's post-hoc tests.

Figure 2

Decreased NeuN positive ⁽⁺⁾ cells induced by 10 μg/kg KA treatment in the CA3 area of the unilateral hippocampus. (A–C) Representative IHC images of NeuN⁺ cells in the unilateral hippocampus. (D) Quantification of NeuN⁺ cells for all treatment groups. Scale bars represent 250 μm. Data represent means ± SEM. ^*P < 0.05, as compared with the control group. ^#P < 0.05, as compared with the 3 μg/kg KA-group. ANOVA tests followed by Duncan's post-hoc tests.

Figure 3

Increased neuronal death induced by 10 μg/kg KA treatment in the CA3 area of the unilateral hippocampus. (A–C) Representative IHC images of FJ-B staining in the CA3 area of the unilateral hippocampus. (D) Quantification of FJ-B ⁽⁺⁾ neurons for all treatment groups. Scale bars represent 50 μm. Data represent means ± SEM. ^*P < 0.05, as compared with the control group. ^#P < 0.05, as compared with the 3 μg/kg KA-group. ANOVA tests followed by Duncan's post-hoc tests.

Figure 4

KA-induced neuronal injury in the hippocampus is associated with autophagic cell death. (A) Western blot analysis with antibodies against Beclin-1, LC3II/LC3I, and ACTIN. (B–C) Comparison of relative expression for Beclin-1 and LC3II/LC3I from the control group vs. the KA treatment groups. All results are expressed as means ± SEM (n = 7 per group). ^*P < 0.05, as compared with the control group; ^#P < 0.05, as compared with the 6 h KA-group; ^$P < 0.05, as compared with the 24 h KA- group. ANOVA tests followed by Duncan's post-hoc tests.

Figure 5

Activation of Iba-1⁽⁺⁾ microglia induced by 10 μg/kg KA treatment in the CA3 area of the unilateral hippocampus. (A–C) Representative IHC images of Iba-1⁽⁺⁾ cells in the CA3 area of the unilateral hippocampus. (D) Quantification of Iba-1⁽⁺⁾ cells for all treatment groups. Scale bars represent 50 μm. Data represent means ± SEM (n = 7 per group). ^*P < 0.05, as compared with the control group. ^#P < 0.05, as compared with the 3 μg/kg KA-group. ANOVA tests followed by Duncan's post-hoc tests.

Figure 6

Activation of GFAP⁺ astrocyte induced by 10 μg/kg KA treatment in the CA3 area of the unilateral hippocampus. (A–C) Representative IHC images of GFAP⁺ cells in the CA3 area of the unilateral hippocampus. (D) Quantification of GFAP⁺ cells for all treatment groups. Scale bars represent 50 μm. Data represent means ± SEM (n = 7 per group). ^*P < 0.05, as compared with the control group. ANOVA tests followed by Duncan's post-hoc tests.

Figure 7

Enhanced immunoreactivity of PECAM⁺ cells induced by 10 μg/kg KA treatment in the CA3 area of the unilateral hippocampus. (A–E) Representative IHC images of PECAM⁺ cells in the CA3 area of the unilateral hippocampus. (F) Quantification of PECAM immunoreactivity for all treatment groups. Scale bars represent 50 μm. Data represent means ± SEM (n = 7 per group). ^*P < 0.05, as compared with the control group. ANOVA tests followed by Duncan's post-hoc tests.

Figure 8

Enhanced activation of ZO-1⁺ cells induced by 10 μg/kg KA treatment in the CA3 area of the unilateral hippocampus. (A–E) Representative IHC images of ZO-1⁺ cells in the CA3 area of the unilateral hippocampus. (F) Quantification of ZO-1 immunoreactivity for all treatment groups. Scale bars represent 50 μm. Data represent means ± SEM (n = 7 per group). ^*P < 0.05, as compared with the control group. ANOVA tests followed by Duncan's post-hoc tests.

Figure 9

Western blot analysis for Claudin-5, ZO-1. (A) Western blot analysis for Claudin-5, ZO-1. (B–C) ROD, a percentage of the immunoblot band. All results are expressed as means ± SEM (n = 7 per group). ^*P < 0.05, as compared with the control group; ^#P < 0.05, as compared with the 6 h KA-group; ^$P < 0.05, as compared with the 24 h KA- group; ^&P < 0.05, as compared with the 48 h KA- group. ANOVA tests followed by Duncan's post-hoc tests.

Figure 10

A schematic depiction of the KA-induced neuronal cell death which is associated with BBB damage and neuronal autophagic injury.